Cell-Penetrating ATF5 Polypeptides and Uses Thereof

ABSTRACT

Provided are cell-penetrating ATF5 polypeptides having a cell-penetrating region and an ATF5 leucine zipper region, compositions comprising the ATF5 polypeptides, and methods of treating a tumor and promoting cytotoxicity in a neoplastic cell using the ATF5 polypeptides.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of priority of U.S. ProvisionalPatent Application No. 62/460,975, filed on Feb. 20, 2017, the entirecontents of which are incorporated by reference.

BACKGROUND

Activating transcription factor 5 (ATF5) is a member of the ATF/CREB(cAMP response element binding protein) family of basic leucine zipperproteins. In the normal developing brain, ATF5 is highly expressed inneural progenitor/neural stem cells where it blocks cell cycle exit andpromotes cell proliferation, thereby inhibiting neurogenesis andgliogenesis. ATF5 downregulation is required to permit neuroprogenitorcell cycle exit and differentiation into either neurons, astrocytes, oroligodendroglia (Greene et al. 2009; Sheng et al. 2010a; Sheng et al.2010b; Arias et al. 2012).

In addition to its role in normal development of the nervous system,ATF5 has also emerged as an oncogenic factor that promotes survival ofgliomas and other tumors. A number of studies have demonstrated thatATF5 is highly expressed in a variety of cancers, includingglioblastoma, breast, pancreatic, lung, and colon cancers, and isessential for glioma cell survival (Monaco et al. 2007; Sheng et al.2010a). In the context of gliomas, overexpression of ATF5 inverselycorrelates with disease prognosis and survival, i.e., glioma patientswith higher ATF5 expression have significantly worse outcomes thanpatients with lower ATF5 expression.

In cancer cells, genes that induce apoptosis are often inactivated ordown-regulated, whereas anti-apoptotic genes are frequently activated oroverexpressed. Consistent with this paradigm, ATF5 upregulatestranscription of anti-apoptotic proteins, including B-cell leukemia 2(Bcl-2) and myeloid cell leukemia 1 (Mcl-1), promoting tumor cellsurvival (Sheng et al., 2010b; Chen et al., 2012).

Based on its role in antagonizing apoptosis and promoting cell survival,combined with high expression levels in cancer cells but not in mostnormal tissues, ATF5 has been identified as an attractive potentialtherapeutic target for cancer therapy. For instance, interference withATF5 activity or expression promotes induction of apoptosis inglioblastoma cells in vitro and in vivo without affecting normalastrocytes (Karpel-Massler et al. 2016).

In terms of structure, ATF5 is a 282-amino acid eukaryotic transcriptionfactor with an N-terminal acidic activation domain and a C-terminalbasic leucine zipper (bZIP) domain. The bZIP domain contains aDNA-binding region and a leucine zipper region. The leucine zipper is acommon structural motif, having a leucine at every seventh amino acid inthe dimerization domain. bZIP transcription factors homo- and/orhetero-dimerize via their leucine zippers to specifically bind to DNA.Wild-type human, rat, and murine ATF5 have the amino acid sequences setforth in NCBI Accession No. NP_001180575, NP_758839, and NP_109618,respectively.

NTAzip-ATF5 (FIG. 1A) is an ATF5 inhibitor in which the ATF5 N-terminalactivation domain is deleted and the DNA binding domain is replaced withan engineered enhanced leucine zipper, i.e., an amphipathic acidica-helical sequence containing heptad repeats with a leucine at everyseventh residue, which extends the wild-type ATF5 leucine zipper region(Angelastro et al. 2003). Cell-penetrating dominant negative ATF5(CP-d/n-ATF5) molecules are improved versions of NTAzip-ATF5, whichcontain a cell-penetrating domain and a truncated ATF5 leucine zipper(relative to wild-type), along with an extended leucine zipper sequence(US 2016/0046686). One example of a CP-d/n ATF5 molecule, ST-2, is shownin 1B.

The present inventors have surprisingly discovered that ST-3 (FIG. 1C),a variant of a CP-d/n-ATF5 molecule that lacks the leucine zipperextension, induces cell death in neoplastic cells. Previous studies havedemonstrated that the enhanced leucine zipper region is required forstability and inhibitory activity of dominant-negative bZIP inhibitors(Krylov et al. 1995; Olive et al. 1997; Moll et al. 2000; Acharya et al.2006). Therefore, the discovery that the ATF5 polypeptides of thepresent invention retain their ability to specifically target and killneoplastic cells in the absence of an extended leucine zipper region wasunexpected.

SUMMARY OF THE INVENTION

Some of the main aspects of the present invention are summarized below.Additional aspects are described in the Detailed Description of theInvention, Examples, Drawings, and Claims sections of this disclosure.The description in each section of this disclosure is intended to beread in conjunction with the other sections. Furthermore, the variousembodiments described in each section of this disclosure can be combinedin various different ways, and all such combinations are intended tofall within the scope of the present invention.

Accordingly, the disclosure provides cell-penetrating polypeptidederivatives of wild-type ATF5 comprising a cell penetrating domain andan ATF leucine zipper domain, wherein an engineered enhanced leucinezipper sequence is absent, compositions and kits comprising the ATF5polypeptides, and methods for treating cancer and inducing cytotoxicityin a neoplastic cell using the ATF polypeptides.

In one aspect, the invention provides a cell-penetrating ATF5polypeptide consisting essentially of a cell-penetrating region and atruncated ATF5 leucine zipper region. In some embodiments, the truncatedATF5 leucine zipper region has an amino acid sequence selected from thegroup consisting of LEGECQGLEARNRELKERAESV (SEQ ID NO: 7) andLEGECQGLEARNRELRERAESV (SEQ ID NO: 8). In some embodiments, thepolypeptide comprises a lactam bridge between positions of SEQ ID NO: 7or SEQ ID NO: 8 selected from the group consisting of: E9 and RΔK13;AΔK10 and E14; and E17 and SΔK21.

In some embodiments, the cell-penetrating region has an amino acidsequence selected from the group consisting of: RQIKIWFQNRRMKWKK (SEQ IDNO: 20), RQLKLWFQNRRMKWKK (SEQ ID NO: 21), YGRKKRRQRRR (SEQ ID NO: 35),and YGRKKRRQRR (SEQ ID NO: 36).

In a further aspect, the invention provides a cell-penetrating ATF5polypeptide comprising an amino acid sequence selected from the groupconsisting of:

(SEQ ID NO: 3) RQIKIWFQNRRMKWKKLEGECQGLEARNRELKERAESV; (SEQ ID NO: 4)RQIKIWFQNRRMKWKKLEGECQGLEARNRELRERAESV; (SEQ ID NO: 5)YGRKKRRQRRRLEGECQGLEARNRELKERAESV; and (SEQ ID NO: 6)YGRKKRRQRRRLEGECQGLEARNRELRERAESV.

In one embodiment, the cell-penetrating ATF5 polypeptide comprises theamino acid sequence RQIKIWFQNRRMKWKKLEGECQGLEARNRELKERAESV (SEQ ID NO:3). In particular embodiments, the polypeptide comprises a lactam bridgebetween positions selected from the group consisting of: K13 and E20;E25 and RΔK29; AΔK26 and E30; and E33 and SΔK37.

In some embodiments, the cell-penetrating ATF5 polypeptide of theinvention comprises an N-terminal acetyl group and/or a C-terminal amidegroup.

In some embodiments, the cell-penetrating ATF5 polypeptide of theinvention is capable of crossing the blood-brain barrier (BBB).

Also provided is a composition comprising a cell-penetrating ATF5polypeptide of the invention. In some embodiments, the composition is apharmaceutical composition. Further provided is a kit comprising acell-penetrating ATF5 polypeptide of the invention, and a nucleic acidmolecule encoding a cell-penetrating ATF5 polypeptide of the invention.

The invention also provides an in vitro or ex vivo method of promotingcytotoxicity in a neoplastic cell, the method comprising contacting theneoplastic cell with a cell-penetrating ATF5 polypeptide of theinvention.

The invention further provides an in vitro or ex vivo method ofpromoting cytotoxicity in a neoplastic cell, the method comprisingintroducing into the neoplastic cell an ATF5 polypeptide consistingessentially of an amino acid sequence selected from the group consistingof LEGECQGLEARNRELKERAESV (SEQ ID NO: 7) and LEGECQGLEARNRELRERAESV (SEQID NO: 8), or a nucleic acid molecule encoding an ATF5 polypeptideconsisting essentially of an amino acid sequence selected from the groupconsisting of LEGECQGLEARNRELKERAESV (SEQ ID NO: 7) andLEGECQGLEARNRELRERAESV (SEQ ID NO: 8).

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A-1C show amino acid sequences of the ATF-5 polypeptidesNTAzip-ATF5 (SEQ ID NO: 1) (FIG. 1A), ST-2 (SEQ ID NO: 2) (FIG. 1B), andST-3 (SEQ ID NO; 3) (FIG. 1C). Where present, the extended leucinezipper domain is underlined, the Penetratin-1 cell penetrating domain isitalicized, and the ATF-5 leucine zipper domain is bolded.

FIG. 2 shows that ST-3 rapidly enters U251 glioblastoma multiforme (GBM)cells and enters the nucleus. Cells were treated with 100 μM ST-2 orST-3 for 1 hour prior to immunofluorescence staining and analysis byscanning cytometry.

FIG. 3A-3C show that ST-3 causes cell death in vitro. ST-3 is morepotent than ST-2 in HL60 promyelocytic leukemia (PML) cells (FIG. 3A)and in U251 GBM cells (FIG. 3B). In T98G glioblastoma cells, viabilitydecreases upon exposure to ST-3 (FIG. 3C). Viability was assessed after72 hours of drug exposure.

FIG. 4 shows that ST-3 caspase activation is enhanced in the presence ofABT-263. Caspase activation was assessed after 24 hours of drugexposure.

FIG. 5 shows cytotoxic activity of ST-3 molecules comprising a lactambridge between K13 and E20 (ST-3-A), between E25 and RΔK29 (ST-3-B),between AΔK26 and E30 (ST-3-C), or between E33 and SΔK37 (ST-3-D).

FIG. 6A-6C show in vivo activity of ST-3. FIG. 6A shows that ST-3represses tumor growth in a U87 subcutaneous tumor model. FIG. 6B showsthat ST-3 delays tumor growth in a patient-derived mesenchymal GBM tumorxenograft model. FIG. 6C shows mean tumor volume in an HL-60subcutaneous tumor model. Nu/Nu mice (n=4) were administered twice dailysubcutaneous injections of ST-3 (25 mg/kg per dose).

DETAILED DESCRIPTION OF THE INVENTION

The compounds and compositions described herein can be used to treatvarious conditions and diseases described herein. The compounds andcompositions also have superior, unexpected properties including, butnot limited to, in vitro and in vivo ability to be cytotoxic toneoplastic cells.

The practice of the present invention will employ, unless otherwiseindicated, conventional techniques of pharmaceutics, formulationscience, protein chemistry, cell biology, cell culture, molecularbiology, transgenic biology, microbiology, recombinant DNA, andimmunology, which are within the skill of the art.

In order that the present invention can be more readily understood,certain terms are first defined. Additional definitions are set forththroughout the disclosure. Unless defined otherwise, all technical andscientific terms used herein have the same meaning as commonlyunderstood by one of ordinary skill in the art to which this inventionis related.

Any headings provided herein are not limitations of the various aspectsor embodiments of the invention, which can be had by reference to thespecification as a whole. Accordingly, the terms defined immediatelybelow are more fully defined by reference to the specification in itsentirety.

All of the references cited in this disclosure are hereby incorporatedby reference in their entireties. In addition, any manufacturers'instructions or catalogues for any products cited or mentioned hereinare incorporated by reference. Documents incorporated by reference intothis text, or any teachings therein, can be used in the practice of thepresent invention. Documents incorporated by reference into this textare not admitted to be prior art.

I. Definitions

The phraseology or terminology in this disclosure is for the purpose ofdescription and not of limitation, such that the terminology orphraseology of the present specification is to be interpreted by theskilled artisan in light of the teachings and guidance.

As used in this specification and the appended claims, the singularforms “a,” “an,” and “the” include plural referents, unless the contextclearly dictates otherwise. The terms “a” (or “an”) as well as the terms“one or more” and “at least one” can be used interchangeably.

Furthermore, “and/or” is to be taken as specific disclosure of each ofthe two specified features or components with or without the other.Thus, the term “and/or” as used in a phrase such as “A and/or B” isintended to include A and B, A or B, A (alone), and B (alone). Likewise,the term “and/or” as used in a phrase such as “A, B, and/or C” isintended to include A, B, and C; A, B, or C; A or B; A or C; B or C; Aand B; A and C; B and C; A (alone); B (alone); and C (alone).

Wherever embodiments are described with the language “comprising,”otherwise analogous embodiments described in terms of “consisting of”and/or “consisting essentially of” are included.

Units, prefixes, and symbols are denoted in their Système Internationalde Unites (SI) accepted form. Numeric ranges are inclusive of thenumbers defining the range, and any individual value provided herein canserve as an endpoint for a range that includes other individual valuesprovided herein. For example, a set of values such as 1, 2, 3, 8, 9, and10 is also a disclosure of a range of numbers from 1-10, from 1-8, from3-9, and so forth. Likewise, a disclosed range is a disclosure of eachindividual value encompassed by the range. For example, a stated rangeof 5-10 is also a disclosure of 5, 6, 7, 8, 9, and 10.

Amino acids are referred to herein by their commonly known three-lettersymbols or by the one-letter symbols recommended by the IUPAC-IUBBiochemical Nomenclature Commission. Unless otherwise indicated, aminoacid sequences are written left to right in amino to carboxyorientation.

The terms “polypeptide,” “peptide,” and “protein” are usedinterchangeably herein to refer to polymers of amino acids of any lengthand their salts. The polymer can be linear or branched, it can comprisemodified amino acids, and can be interrupted non-amino acids. The termsalso encompass an amino acid polymer that has been modified naturally orby intervention; for example, disulfide bond formation, lactam bridgeformation, glycosylation, lipidation, acetylation, acylation, amidation,phosphorylation, or other manipulation or modification, such asconjugation with a labeling component or addition of a protecting group.Also included within the definition are, for example, polypeptidescontaining one or more analogs of an amino acid (including, for example,unnatural amino acids, etc.), as well as other modifications known inthe art. In certain embodiments, the polypeptides can occur as singlechains, covalent dimers, or non-covalent associated chains. Polypeptidescan also contain one or more bridges or cross-links within the sequence.The spacing of cross-links between amino acids can be, for example, 3,4,or 7 residues apart, preferably 3 or 4 residues apart. In some cases,the internal cross-link replaces the side chains of cross-linkedresidues. In some cases, the amino acid side chains are replaced withhydrocarbon chains. In some cases, the original amino acid(s) in thesequence are substituted with other amino acids so that a link can bemade, for example, by bridging free amino and free carboxyl sidechaingroups via a lactam. Substitution is denoted herein by a Δ between theoriginal amino acid and the substituted amino acid. Formation of thecyclic compounds can be achieved by treatment with a dehydrating agent,with suitable protection if needed. The open chain (linear form) tocyclic form reaction can involve intramolecular-cyclization.

The term “conservative substitution” as used herein denotes that one ormore amino acids are replaced by another, biologically similar residue.Examples include substitution of amino acid residues with similarcharacteristics, e.g., small amino acids, acidic amino acids, polaramino acids, basic amino acids, hydrophobic amino acids, and aromaticamino acids. For further information concerning phenotypically silentsubstitutions in peptides and proteins, see, for example, Bowie et. al.,Science 247:1306-1310 (1990). In the scheme below, conservativesubstitutions of amino acids are grouped by physicochemical properties;I: neutral and/or hydrophilic, II: acids and amides, III: basic, IV:hydrophobic, V: aromatic, bulky amino acids.

In the scheme below, conservative substitutions of amino acids aregrouped by physicochemical properties; VI: neutral or hydrophobic, VII:acidic, VIII: basic, IX: polar, X: aromatic.

Methods of identifying conservative nucleotide and amino acidsubstitutions which do not affect protein function are well-known in theart (see, e.g., Brummell et al., Biochem. 32 :1180-1187 (1993);Kobayashi et al., Protein Eng. 12(10):879-884 (1999); and Burks et al.,Proc. Natl. Acad. Sci. U.S.A. 94:412-417 (1997)).

The terms “identical” or percent “identity” in the context of two ormore nucleic acids or polypeptides, refers to two or more sequences orsubsequences that are the same or have a specified percentage ofnucleotides or amino acid residues that are the same, when compared andaligned (introducing gaps, if necessary) for maximum correspondence, notconsidering any conservative amino acid substitutions as part of thesequence identity. The percent identity can be measured using sequencecomparison software or algorithms, or by visual inspection. Variousalgorithms and software are known in the art that can be used to obtainalignments of amino acid or nucleotide sequences.

One such non-limiting example of a sequence alignment algorithm isdescribed in Karlin et al., Proc. Natl. Acad. Sci., 87:2264-2268 (1990),as modified in Karlin et al., Proc. Natl. Acad. Sci., 90:5873-5877(1993), and incorporated into the NBLAST and XBLAST programs (Altschulet al., Nucleic Acids Res., 25:3389-3402 (1991)). In certainembodiments, Gapped BLAST can be used as described in Altschul et al.,Nucleic Acids Res. 25:3389-3402 (1997). BLAST-2, WU-BLAST-2 (Altschul etal., Methods in Enzymology, 266:460-480 (1996)), ALIGN, ALIGN-2(Genentech, South San Francisco, Calif.) or Megalign (DNASTAR) areadditional publicly available software programs that can be used toalign sequences. In certain embodiments, the percent identity betweentwo nucleotide sequences is determined using the GAP program in the GCGsoftware package (e.g., using a NWSgapdna.CMP matrix and a gap weight of40, 50, 60, 70, or 90 and a length weight of 1, 2, 3, 4, 5, or 6). Incertain alternative embodiments, the GAP program in the GCG softwarepackage, which incorporates the algorithm of Needleman and Wunsch (J.Mol. Biol. (48):444-453 (1970)), can be used to determine the percentidentity between two amino acid sequences (e.g., using either a BLOSUM62 matrix or a PAM250 matrix, and a gap weight of 16, 14, 12, 10, 8, 6,or 4 and a length weight of 1, 2, 3, 4, 5). Alternatively, in certainembodiments, the percent identity between nucleotide or amino acidsequences is determined using the algorithm of Myers and Miller (CABIOS4:11-17 (1989)). For example, the percent identity can be determinedusing the ALIGN program (version 2.0) and using a PAM120 with residuetable, a gap length penalty of 12 and a gap penalty of 4. One skilled inthe art can determine appropriate parameters for maximal alignment byparticular alignment software. In certain embodiments, the defaultparameters of the alignment software are used. Other resources forcalculating identity include methods described in ComputationalMolecular Biology (Lesk ed., 1988); Biocomputing: Informatics and GenomeProjects (Smith ed., 1993); Computer Analysis of Sequence Data, Part 1(Griffin and Griffin eds., 1994); Sequence Analysis in Molecular Biology(G. von Heinje, 1987); Sequence Analysis Primer (Gribskov et al. eds.,1991); and Carillo et al., SIAM J. Applied Math., 48:1073 (1988).

In certain embodiments, the percentage identity “X” of a first aminoacid sequence to a second sequence amino acid is calculated as100×(Y/Z), where Y is the number of amino acid residues scored asidentical matches in the alignment of the first and second sequences (asaligned by visual inspection or a particular sequence alignment program)and Z is the total number of residues in the second sequence. If thelength of a first sequence is longer than the second sequence, thepercent identity of the first sequence to the second sequence will behigher than the percent identity of the second sequence to the firstsequence.

A “polynucleotide,” as used herein can include one or more “nucleicacids,” “nucleic acid molecules,” or “nucleic acid sequences,” andrefers to a polymer of nucleotides of any length, and includes DNA andRNA. The polynucleotides can be deoxyribonucleotides, ribonucleotides,modified nucleotides or bases, and/or their analogs, or any substratethat can be incorporated into a polymer by DNA or RNA polymerase. Apolynucleotide can comprise modified nucleotides, such as methylatednucleotides and their analogs. The preceding description applies to allpolynucleotides referred to herein, including RNA and DNA.

An “isolated” molecule is one that is in a form not found in nature,including those which have been purified.

A “label” is a detectable compound that can be conjugated directly orindirectly to a molecule, so as to generate a “labeled” molecule. Thelabel can be detectable on its own (e.g., radioisotope labels orfluorescent labels) or can catalyze chemical alteration of a substratecompound or composition that is detectable (e.g., an enzymatic label).

The term “operably linked” refers to the positioning of two or moremolecules or sequences in a relationship permitting them to function intheir intended manner. For example, a control sequence operably linkedto a coding sequence is positioned in such a way that expression of thecoding sequence is achieved under conditions compatible with the controlsequence. Operable linkage can be covalent or non-covalent, dependingupon the identity and intended function of the operably linkedcomponents.

“Binding affinity” generally refers to the strength of the sum total ofnon-covalent interactions between a single binding site of a moleculeand its binding partner (e.g., a receptor and its ligand, an antibodyand its antigen, two monomers that form a dimer, etc.). Unless indicatedotherwise, as used herein, “binding affinity” refers to intrinsicbinding affinity which reflects a 1:1 interaction between members of abinding pair. The affinity of a molecule X for its partner Y cangenerally be represented by the dissociation constant (K_(D)). Affinitycan be measured by common methods known in the art, including thosedescribed herein. Low-affinity binding partners generally bind slowlyand tend to dissociate readily, whereas high-affinity binding partnersgenerally bind faster and tend to remain bound longer.

The affinity or avidity of a molecule for its binding partner can bedetermined experimentally using any suitable method known in the art,e.g., flow cytometry, enzyme-linked immunosorbent assay (ELISA), orradioimmunoassay (MA), or kinetics (e.g., KINEXA® or BIACORE™ or OCTET®analysis). Direct binding assays as well as competitive binding assayformats can be readily employed. (See, e.g., Berzofsky et al.,“Antibody-Antigen Interactions,” In Fundamental Immunology, Paul, W. E.,ed., Raven Press: New York, N.Y. (1984); Kuby, Immunology, W. H. Freemanand Company: New York, N.Y. (1992)). The measured affinity of aparticular binding pair interaction can vary if measured under differentconditions (e.g., salt concentration, pH, temperature). Thus,measurements of affinity and other binding parameters (e.g., K_(D) orKd, K_(on), K_(off)) are made with standardized solutions of bindingpartners and a standardized buffer, as known in the art.

An “active agent” is an ingredient that is intended to furnishpharmacological activity or other direct effect in the diagnosis, cure,mitigation, treatment, or prevention of disease, or to affect thestructure or any function of the human body. The active agent can be inassociation with one or more other ingredients, and can be, but is notnecessarily, in a finished dosage form. The terms “active agent” and“drug substance” are used interchangeably herein.

An “effective amount” of an active agent is an amount sufficient tocarry out a specifically stated purpose.

The term “pharmaceutical composition” refers to a preparation that is insuch form as to permit the biological activity of the active ingredientto be effective and which contains no additional components that areunacceptably toxic to a subject to which the composition would beadministered. Such composition can be sterile and can comprise apharmaceutically acceptable carrier, such as physiological saline.Suitable pharmaceutical compositions can comprise one or more of abuffer (e.g. acetate, phosphate or citrate buffer), a surfactant (e.g.polysorbate), a stabilizing agent (e.g. polyol or amino acid), apreservative (e.g. sodium benzoate), a penetration enhancer, anabsorption promoter to enhance bioavailability and/or other conventionalsolubilizing or dispersing agents. Choice of excipients depends upondosage form, the active agent to be delivered, and the disease ordisorder to be treated or prevented.

A “subject” or “individual” or “animal” or “patient” or “mammal,” is anysubject, particularly a mammalian subject, for whom diagnosis,prognosis, or therapy is desired. Mammalian subjects include humans,domestic animals, farm animals, sports animals, and laboratory animalsincluding, e.g., humans, non-human primates, canines, felines, porcines,bovines, equines, rodents, including rats and mice, rabbits, etc.

Terms such as “treating” or “treatment” or “to treat” or “alleviating”or “to alleviate” refer to therapeutic measures that cure, slow down,lessen symptoms of, and/or halt progression of an undesiredphysiological condition, a diagnosed pathologic condition, disease, ordisorder. Thus, those in need of treatment include those already withthe disorder. In certain embodiments, a subject is successfully“treated” for a disease or disorder if the patient shows, e.g., total,partial, or transient alleviation or elimination of symptoms associatedwith the condition, disease, or disorder; diminishment of the extent ofthe condition, disease, or disorder; stabilization (i.e., not worsening)of the condition, disease, or disorder; delay in onset or slowing ofprogression of the condition, disease or disorder; amelioration of thecondition, disease, or disorder, including partial or total remission;and/or prolonging survival, as compared to expected survival if notreceiving treatment. In the context of the present invention, reductionof tumor volume is one example of treatment. Tumor volume can bemonitored by any known method, including magnetic resonance imaging,computed tomographic imaging, positron emission tomography, sonography,mammography, and physical measurement.

“Prevent” or “prevention” refer to prophylactic or preventative measuresthat prevent and/or slow the development of a targeted pathologiccondition or disorder. Thus, those in need of prevention include thoseprone to have or susceptible to the disorder. In certain embodiments, adisease or disorder is successfully prevented if the patient develops,transiently or permanently, e.g., fewer or less severe symptomsassociated with the disease or disorder, or a later onset of symptomsassociated with the disease or disorder, than a patient who has not beensubject to the methods of the invention.

The terms “inhibit,” “block,” and “suppress” are used interchangeablyand refer to any statistically significant decrease in occurrence oractivity, including full blocking of the occurrence or activity. Forexample, “inhibition” can refer to a decrease of about 10%, 20%, 30%,40%, 50%, 60%, 70%, 80%, 90% or 100% in activity or occurrence. An“inhibitor” is a molecule, factor, or substance that produces astatistically significant decrease in the occurrence or activity of aprocess, pathway, or molecule.

In functional assays, “EC₅₀” is the concentration that reduces abiological response by 50% of its maximum. In the case of ATF5polypeptides, EC₅₀ is measured as the concentration that reduces cellviability by 50% of its maximum. EC₅₀ can be calculated by any number ofmeans known in the art.

A “neoplastic cell” or “neoplasm” typically has undergone some form ofmutation/transformation, resulting in abnormal growth as compared tonormal cells or tissue of the same type. Neoplasms include morphologicalirregularities, as well as pathologic proliferation. Neoplastic cellscan be benign or malignant. Malignant neoplasms, i.e., cancers, aredistinguished from benign in that they demonstrate loss ofdifferentiation and orientation of cells, and have the properties ofinvasion and metastasis.

A “solid tumor” is a mass of neoplastic cells. A “liquid tumor” or a“hematological malignancy” is a blood cancer of myeloid or lymphoidlineage. Hematological malignancies include leukemias, lymphomas, andmyelomas. Examples of leukemia include acute lymphoblastic leukemia(ALL), acute myelogenous leukemia (AML), chronic lymphocytic leukemia(CLL), chronic myelogenous leukemia (CML), and acute monocytic leukemia(AMoL). Examples of lymphoma include Hodgkin's lymphoma andnon-Hodgkin's lymphoma. Multiple myeloma is an example of a myeloma.

II. ATF5 Polypeptides and Compositions Cell-Penetrating Peptides

The ATF5 polypeptides of the present invention preferably comprise acell-penetrating domain or cell-penetrating peptide (CPP). The terms“cell-penetrating domain,” “cell-penetrating region,” and“cell-penetrating peptide” are used interchangeably herein.

CPPs are short (typically about 6-40 amino acids) peptides that are ableto cross cell membranes. Peptides referred to as nuclear localizationsequences are a subset of CPPs. Many CPPs are capable of crossing theblood-brain barrier (BBB). In some embodiments, the CPP is 7, 8, 9, 10,11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28,29, 30, 31, 32, 33, 34, 35, 36, 37, 38, or 39 amino acids in length,including ranges having any of those lengths as endpoints, for example,10-30 amino acids. CPPs are typically water soluble, cationic oramphipathic, and rich in basic amino acids (e.g., lysine and/or arginineresidues). CPPs can also be positively charged amphipathic peptides, orpeptides that are hydrophobic, containing only apolar residues with lownet charge or hydrophobic amino acid groups. CPPs have the ability totransport covalently or non-covalently linked molecular cargo, such aspolypeptides, polynucleotides, and nanoparticles, across cell membranesand the BBB. The translocation can be endocytotic or energy-independent(i.e., non-endocytotic) via translocation. Numerous CPPs are describedand characterized in the literature (see, e.g., Handbook ofCell-Penetrating Peptides (2d ed. Ulo Langel ed., 2007); Hervé et al.2008; Heitz et al. 2009; Munyendo et al. 2012; Zou et al. 2013;Krautwald et al. 2016). A curated database of CPPs is maintained atcrdd.osdd.net/raghava/cppsite (Gautam et al. 2012).

Non-limiting examples of CPPs suitable for use in the present inventioninclude peptides derived from proteins, such as from Drosophilaantennapedia transcription factor (Penetratin and its derivatives RL-16and EB1) (Derossi et al. 1998; Thorén et al. 2000; Lundberg et al. 2007;Alves et al. 2008); from HIV-1 trans-activator of transcription (Tat)(Vivès et al. 1997; Hällbrink et al. 2001); from rabies virusglycoprotein (RVG) (Kumar et al. 2007); from herpes simplex virus VP22(Elliott et al. 1997); from antimicrobial protegrin 1 (SynB) (Rousselleet al. 2001), from rat insulin 1 gene enhancer protein (pIS1) (Kilk etal. 2001; Magzoub et al. 2001); from murine vascular endothelialcadherein (pVEC) (Elmquist et al. 2001); from human calcitonin (hCT)(Schmidt et al. 1998); and from fibroblast growth factor 4 (FGF4) (Jo etal. 2005). CPPs suitable for use in the invention also include syntheticand chimeric peptides, such as Transportan (TP) and its derivatives(Pooga et al. 1998; Soomets et al. 2000); membrane translocatingsequences (MTSs) (Brodsky et al. 1998; Lindgren et al. 2000; Zhao et al.2001), such as the MPS peptide (also known as fusion sequence-basedpeptide or FBP) (Chaloin et al. 1998); sequence signal-based peptide(SBP) (Chaloin et al. 1997); model amphipathic peptide (MAP) (Oehlke etal. 1998; Scheller et al. 1999; Hällbrink et al. 2001), translocatingpeptide 2 (TP2) (Cruz et al. 2013), MPG (Morris et al. 1997; Kwon et al.2009), Pep-1 (Morris et al. 2001; Muñoz-Morris et al. 2007), andpoly-arginine (e.g., R₇-R₁₂) (Mitchell et al. 2000; Wender et al. 2000;Futaki et al. 2001; Suzuki et al. 2002). Representative but non-limitingCPP sequences are shown in Table 1.

TABLE 1 Peptide Sequence C. elegans SDC3 FKKFRKF (SEQ ID NO: 9) CADY-KGLWRALWRLLRSLWRLLWK (SEQ ID NO: 10) EB1 CPP LIRLWSHLIHIWFQNRRLKWKKK(SEQ ID NO: 11) FBP CPP GALFLGWLGAAGSTMGAWSQPKKKRKV (SEQ ID NO: 12)FGF4 CPP AAVALLPAVLLALLAP (SEQ ID NO: 13) HATF3 ERKKRRRE (SEQ ID NO: 14)hCT CPP LGTYTQDFNKTFPQTAIGVGAP (SEQ ID NO: 15) MAP CPPKLALKLALKALKAALKLA (SEQ ID NO: 16) MPG CPP GLAFLGFLGAAGSTMGAWSQPKKKRKV(SEQ ID NO: 17) NF-κB VQRKRQKLMP (SEQ ID NO: 18) OCT-6 GRKRKKRT(SEQ ID NO: 19) Penetratin CPP RQIKIWFQNRRMKWKK (SEQ ID NO: 20)Penetratin CPP RQLKLWFQNRRMKWKK variant 1 (SEQ ID NO: 21) Penetratin CPPREIKIWFQNRRMKWKK variant 2 (SEQ ID NO: 22) Pep-1 CPPKETWWETWWTEWSQPKKRKV (SEQ ID NO: 23) pIsl CPP PVIRVWFQNKRCKDKK(SEQ ID NO: 24) Poly-Arg CPP RRRRRR(R)₁₋₆  (SEQ ID NO: 25) pVEC CPPLLIILRRRIRKQAHAH (SEQ ID NO: 26) RL-16 CPP RRLRRLLRRLLRRLRR(SEQ ID NO: 27) RVG CPP RVGRRRRRRRRR (SEQ ID NO: 28) R₆W₃ CPP RRWWRRWRR(SEQ ID NO: 29) SBP CPP MGLGLHLLVLAAALQGAWSQPKKKRKV (SEQ ID NO: 30) SV40PKKKRKV (SEQ ID NO: 31) SynB1 CPP RGGRLSYSRRFSTSTGR (SEQ ID NO: 32)SynB3 CPP RRLSYSRRRF (SEQ ID NO: 33) SynB5 CPP RGGRLAYLRRRWAVLGR(SEQ ID NO: 34) Tat⁴⁷⁻⁵⁷ CPP YGRKKRRQRRR (SEQ ID NO: 35) Tat⁴⁷⁻⁵⁶ CPPYGRKKRRQRR (SEQ ID NO: 36) Tat⁴⁸⁻⁵⁶ CPP GRKKRRQRR (SEQ ID NO: 37)Tat⁴⁸⁻⁶⁰ CPP GRKKRRQRRRPPQ (SEQ ID NO: 38) TCF1-α GKKKKRKREKL(SEQ ID NO: 39) TFIIE-β SKKKKTKV (SEQ ID NO: 40) TP CPPGWTLNSAGYLLGKINLKALAALAKKIL (SEQ ID NO: 41) TP10 CPPAGYLLGKINLKALAALAKKIL (SEQ ID NO: 42) TP2 CPP PLIYLRLLRGQF(SEQ ID NO: 43) VP22 CPP DAATATRGRSAASRPTQRPRAPARSASRPRRPVQ(SEQ ID NO: 44)

Because the function of CPPs depends on their physical characteristicsrather than sequence-specific interactions, they can have the reversesequence as those provided in Table 1 and/or known in the art. Variantsof these sequences with one or more amino acid additions, deletions,and/or conservative substitutions that retain the ability to cross cellmembranes and/or the BBB are also suitable for use in the invention. TheATF5 polypeptides of the invention can include a cell-penetrating domainhaving at least about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%,80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, or 99% identity to the exemplary sequencesprovided in Table 1. The effect of the amino acid addition(s),deletion(s), and/or substitution(s) on the ability of the CPP to mediatecell penetration can be tested using routine methods known in the art.

ATF5 Polypeptides

In one aspect, the invention provides cell-penetrating ATF5 polypeptideshaving a cell-penetrating region and a truncated ATF5 leucine zipperregion, relative to wild-type ATF5. The ATF-5 polypeptides of theinvention lack an extended leucine zipper region, such as that which ispresent in NTAzip-ATF5 (FIG. 1A) and ST-2 (FIG. 1B). As used herein, theterms “extended leucine zipper,” “leucine zipper extension,” and“enhanced leucine zipper” refer to a peptide having from one to fourleucine heptads, i.e., Leu-(X)₆ (SEQ ID NO: 45), which sequence is not awild-type ATF5 leucine zipper sequence. The cell-penetrating ATF5polypeptides of the invention are capable of crossing a cell membrane,by virtue of their cell-penetrating region, and inhibiting ATF5 activityin the cell. In some embodiments, the cell-penetrating ATF5 polypeptidecan cross the BBB. In some embodiments, the ATF5 polypeptide can affectpathways involved in apoptosis. ATF5 activity can be assessed by any ofseveral assays known in the art, including the cell-kill assay describedherein. ATF5 activity can also be assessed by its ability to bind to thecAMP-response element (CRE).

The “ATF5 leucine zipper region” is a truncated sequence derived fromthe wild-type ATF5 leucine zipper region. The term is used to refer onlyto sequence, and not necessarily to secondary structure. The truncatedATF5 leucine zipper region can have, for example, an amino acid sequenceselected from the group consisting of LEGECQGLEARNRELKERAESV (SEQ ID NO:7) and LEGECQGLEARNRELRERAESV (SEQ ID NO: 8). ATF5 polypeptides of theinvention include polypeptides consisting essentially of the ATF5leucine zipper region. Conservative amino acid substitutions areincluded in the scope of these sequences.

The cell-penetrating region is operably linked to the ATF5 leucinezipper region. In some embodiments, the cell-penetrating region iscovalently linked to the ATF5 leucine zipper region, for example, via apeptide bond, a disulfide bond, a thioether bond, or a linker known inthe art. Exemplary linkers include, but are not limited to, asubstituted alkyl or a substituted cycloalkyl. A cell-penetrating regionand an ATF5 leucine zipper region linked directly by an amide bond maybe referred to as a “fusion.” The cell-penetrating region can be linkedto the N-terminus or the C-terminus of the ATF5 leucine zipper region,or via a residue side chain.

Cell-penetrating ATF5 polypeptides of the invention can comprise anycombination of cell-penetrating and ATF5 leucine zipper domainsdisclosed herein. Non-limiting examples of such polypeptides include

RQIKIWFQNRRMKWKKLEGECQGLEARNRELKERAESV (SEQ ID NO: 3);RQIKIWFQNRRMKWKKLEGECQGLEARNRELRERAESV (SEQ ID NO: 4);YGRKKRRQRRRLEGECQGLEARNRELKERAESV (SEQ ID NO: 5); and

YGRKKRRQRRRLEGECQGLEARNRELRERAESV (SEQ ID NO: 6). In one embodiment, theATF5 polypeptide is ST-3 (FIG. 1C), having the sequenceRQIKIWFQNRRMKWKKLEGECQGLEARNRELKERAESV (SEQ ID NO: 3).

ATF5 polypeptides of the invention include polypeptides having at leastabout 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to those sequencesdisclosed herein.

ATF5 polypeptides can optionally include an N-terminal acetyl groupand/or a C-terminal amide group. ATF5 polypeptides of the invention canoptionally include one or more internal cyclizations, such as lactambridges or hydrocarbon “staples.” A lactam bridge is preferably, but notnecessarily, created between side chains spaced four amino acid residuesapart (BxxxB).

ATF5 polypeptides of the invention can optionally include one or moreepitope and/or affinity tags, such as for purification or detection.Non-limiting examples of such tags include FLAG, HA, His, Myc, GST, andthe like. ATF5 polypeptides of the invention can optionally include oneor more labels.

In certain aspects, the invention provides a composition, e.g., apharmaceutical composition, comprising an ATF5 polypeptide of theinvention, optionally further comprising one or more carriers, diluents,excipients, or other additives. The cell-penetrating ATF5 polypeptidesof the invention can be formulation for enteral, parenteral,transdermal, or transmucosal administration, as discussed below.Pharmaceutical compositions can be in numerous dosage forms, forexample, tablet, capsule, liquid, solution, softgel, suspension,emulsion, syrup, elixir, tincture, film, powder, ointment, paste, cream,lotion, gel, mousse, foam, lacquer, spray, aerosol, inhaler, nebulizer,ophthalmic drops, patch, suppository, and/or enema.

Also within the scope of the invention are kits comprising the ATF5polypeptides and compositions as provided herein and, optionally,instructions for use. The kit can further contain at least oneadditional reagent, and/or one or more additional active agent. Kitstypically include a label indicating the intended use of the contents ofthe kit. The term “label” in this context includes any writing orrecorded material supplied on or with the kit, or that otherwiseaccompanies the kit.

ATF5 polypeptides of the invention can be chemically synthesized, forexample, using solid-phase peptide synthesis or solution-phase peptidesynthesis, or can be expressed using recombinant methods. Synthesis mayoccur as fragments of the peptide that are subsequently combined, eitherchemically or enzymatically. Accordingly, also provided are nucleic acidmolecules encoding ATF5 polypeptides of the invention. Such nucleicacids can be constructed by chemical synthesis using an oligonucleotidesynthesizer. Nucleic acid molecules of the invention can be designedbased on the amino acid sequence of the desired ATF5 polypeptide andselection of those codons that are favored in the host cell in which therecombinant ATF5 polypeptide will be produced. Standard methods can beapplied to synthesize a nucleic acid molecule encoding an ATF5polypeptide of interest.

Once prepared, the nucleic acid encoding a particular ATF5 polypeptideof the invention can be inserted into an expression vector and operablylinked to an expression control sequence appropriate for expression ofthe polypeptide in a desired host or in a target cell, such as aneoplastic cell. In order to obtain high expression levels of the ATF5polypeptide, the nucleic acid can be operably linked to or associatedwith transcriptional and translational expression control sequences thatare functional in the chosen expression host or target cell.

A wide variety of expression cell/vector combinations can be employed byone skilled in the art. Useful expression vectors for eukaryotic cellsinclude, for example, vectors comprising expression control sequencesfrom SV40, bovine papilloma virus, adenovirus, and cytomegalovirus.Useful expression vectors for bacterial cells include known bacterialplasmids, such as plasmids from E. coli, including pCR1, pBR322, pMB9and their derivatives, wider host range plasmids, such as M13, andfilamentous single-stranded DNA phages.

Suitable host cells include prokaryotes, yeast, insect, or highereukaryotic cells under the control of appropriate promoters. Prokaryotesinclude gram negative or gram positive organisms, for example E. coli orbacilli. Higher eukaryotic cells can be established or cell lines ofmammalian origin, examples of which include Pichia pastoris, 293 cells,COS-7 cells, L cells, C127 cells, 3T3 cells, Chinese hamster ovary (CHO)cells, HeLa cells, and BHK cells. Cell-free translation systems can alsobe employed.

III. Methods of Use

The ATF5 polypeptides of the invention can be used to promotecytotoxicity in a neoplastic cell. Cytotoxicity can be measured by knownassays, including the cell kill assay described herein. The ATF5polypeptides of the invention can also be used in methods of treatment,in particular, for the treatment of tumors and hematologicalmalignancies.

Thus, in one aspect, the invention provides a method of treating a solidor liquid tumor in a subject, the method comprising administering to thesubject an effective amount of a cell-penetrating ATF5 polypeptide ofthe invention. The invention also provides a cell-penetrating ATF5polypeptide for use in treating a solid or liquid tumor. The inventionfurther provides the use of a cell-penetrating ATF5 polypeptide for themanufacture of a medicament for the treatment of a solid or liquidtumor. In some embodiments, the tumor is malignant. In some embodiments,the tumor is a hematological malignancy. In particular, the inventionprovides a cell-penetrating ATF5 polypeptide for use in treating ahematological malignancy. In addition, the invention provides a methodof treating a hematological malignancy in a subject, the methodcomprising administering to the subject an effective amount of acell-penetrating ATF5 polypeptide of the invention. In one embodiment,the hematological malignancy is leukemia. In a particular embodiment,the leukemia is acute myelogenous leukemia.

The invention additionally provides a method of promoting cytotoxicityin a neoplastic cell, the method comprising contacting the neoplasticcell with a cell-penetrating ATF5 polypeptide of the invention. Further,the invention provides a cell-penetrating ATF5 polypeptide of theinvention for use in promoting cytotoxicity in a neoplastic cell. Theinvention also provides the use of a cell-penetrating ATF5 polypeptidefor the manufacture of a medicament for promoting cytotoxicity in aneoplastic cell.

The invention provides a method of promoting cytotoxicity in aneoplastic cell, the method comprising introducing into a neoplasticcell a nucleic acid molecule encoding an ATF5 polypeptide of theinvention. Also provided, is a nucleic acid molecule encoding an ATF5polypeptide of the invention for use in promoting cytotoxicity in aneoplastic cell. Further provided is the use of a nucleic acid moleculeencoding an ATF5 polypeptide of the invention for the manufacture of amedicament for promoting cytotoxicity in a neoplastic cell. In someembodiments, the ATF5 polypeptide comprises a cell-penetrating region.In some embodiments, the ATF5 polypeptide consists essentially of anATF5 leucine zipper region. In certain embodiments, the ATF5 polypeptideconsists essentially of an amino acid sequence selected from the groupconsisting of LEGECQGLEARNRELKERAESV (SEQ ID NO: 7) andLEGECQGLEARNRELRERAESV (SEQ ID NO: 8).

In certain embodiments, the neoplastic cell is in vitro or ex vivo. Insome such embodiments, the neoplastic cell can be, for example, abladder, blood, breast, cervical, colon, endometrial, endothelial,esophageal, gastric, intestinal, kidney, larynx, liver, lung, lymphnode, mouth, neural, ovarian, pancreatic, parotid, pharynx, prostate,skin, testicular, tongue, or uterine cell. In some such embodiments, theneoplastic cell can be, for example, a glioma, a medulloblastoma, or aneuroblastoma cell, in particular, an astrocytoma or a glioblastomacell.

ATF5 has been shown to play a role in promoting radioresistance andmalignant phenotypes in A549 human lung adenocarcinoma cells.ATF5-enhanced radioresistance was shown to occur through increasedprotein expression in the G1-S phase, thereby promoting cell cycleprogression and preventing cell senescence. ATF5 was further shown toinduce invasiveness of A549 cancer cells by promoting expression of thecell-matrix adhesion protein, integrin β1, through stabilization andalso through inhibition of myosin regulatory light chain (MRLC)diphosphorylation (Ishihara et al. 2015). These data suggest that ATF5functions as one of the key molecules in oncogenic resistance toradiotherapy. Accordingly, in one aspect, the invention provides amethod of treating a tumor in a subject comprising administering acell-penetrating ATF5 polypeptide of the invention in combination withradiation therapy.

A cell-penetrating ATF5 polypeptide of the invention can be administeredin combination with one or more additional active agents, for exampleone or more active agents used to treat cancer. The ATF5 polypeptide andadditional active agent(s) can act additively or synergistically. Theadditional active agent can be a Bcl-2 inhibitor, such as a BH3 mimetic.Non-limiting examples of BH3 mimetics include ABT-263 (Navitoclax),ABT-199 (Venetoclax), S-055746, and PNT-2258. The additional activeagent can be one or more chemotherapeutic agents and/or targetedtherapeutic agents, including biologic agents. Examples ofchemotherapeutic agents include actinomycin, azacitidine, azathioprine,bleomycin, bortezomib, carboplatin, capecitabine, carmustine, cisplatin,chlorambucil, cyclophosphamide, cytarabine, daunorubicin, docetaxel,doxifluridine, doxorubicin, epirubicin, epothilone, etoposide,fluorouracil, gemcitabine, hydroxyurea, idarubicin, imatinib,irinotecan, lomustine, mechlorethamine, mercaptopurine, methotrexate,mitoxantrone, oxaliplatin, paclitaxel, pemetrexed, temozolomide,teniposide, tioguanine, topotecan, tretinoin, valrubicin, vinblastine,vincristine, vindesine, and vinorelbine. Examples of targetedtherapeutic agents include bevacizumab, pazopanib, poly(ADP-ribose)polymerase (PARP) inhibitors, such as olaparib, tamoxifen, andvintafolide. Combined therapies can be sequential or concurrent.

Administration of the cell-penetrating ATF5 polypeptides of theinvention can be via any suitable route determined by the ordinarilyskilled artisan. Exemplary routes of administration include enteral,parenteral, transdermal, and transmucosal. Enteral routes involve thealimentary canal, and include oral, sublingual, buccal, and rectaladministration. Parenteral routes do not involve the alimentary canal,and may include administration by injection. Examples of parenteraladministration include intracerebral, intracranial, intramuscular,intraperitoneal, intrathecal, intratumoral, intravenous, subcutaneous,and ocular. Transdermal administration involves application of theactive agent to the skin of the subject. The transmucosal route involvesabsorption of the active agent by the mucous membranes of the subject,for example, via the nasal, sinus, bronchial (e.g., via inhalation), orvaginal mucosa. Device-mediated administration, such as by osmotic pump,cartridge, or micro pump is also included.

EXAMPLES

Embodiments of the present disclosure can be further defined byreference to the following non-limiting examples. It will be apparent tothose skilled in the art that many modifications, both to materials andmethods, can be practiced without departing from the scope of thepresent disclosure.

Example 1 Cytoplasmic and Nuclear Penetration of ST-3 in GlioblastomaMultiforme (GBM) Cells

ST-2 (FIG. 1B) is a 67-amino-acid polypeptide containing an N-terminalcell penetrating domain, followed by an engineered leucine zipperextension, and a rat ATF5 bZIP domain truncated after the first valine.It has been demonstrated previously that systemically administered ST-2can cross the blood-brain barrier, enter cells, and selectively triggerthe apoptotic death of neoplastic cells in a mouse glioma model (U.S.Pat. No. 9,758,555). ST-3 (FIG. 1C) is a 38-amino-acid polypeptidecontaining an N-terminal cell penetrating domain, followed by the humanATF5 bZIP domain truncated after the first valine. Prior to the presentinvention, the leucine zipper extension region, which ST-2 contains andST-3 lacks, was thought to be required for activity of the molecule.

All cellular penetration assays were performed in U251 cells.Immunofluorescence detection of ST-3 in U251 cells allow forquantitation of cellular and nuclear penetration. U251 cells were seededat a density of 5×10³ in a 96-well plate and cultured at 37° C. in 5%CO₂. At approximately 12-24 hours post-seeding, U251 cells were treatedwith ST-3 (or ST-2) for 1-4 hours. Following extensive washing in PBS,cells were fixed in 1% formaldehyde for 15 minutes at room temperature(RT), permeabilized with 0.1% Triton-X-100 for 15 minutes at RT, andblocked with 1% BSA in PBS for 30 minutes at RT. Fixed cells wereincubated with SPTX-001 rabbit polyclonal anti-ST-3 antibody (1:2000dilution) overnight at 4° C. or for 90 minutes at RT, followed byincubation with FITC-conjugated goat ant-rabbit secondary antibody(1:500-5000 dilution) for 45-60 minutes at RT. Nuclei were visualizedusing DAPI staining (2.85 μg/mL) for 15 minutes at RT. ST-3-positivecells were measured by a CompuCyte iCys scanning cytometer withfour-color analysis. Analysis supported gating a quantification ofST-3-positive population.

ST-3 (100 μM) penetrated 100% of cytoplasmic membranes and about 84% ofnuclear membranes in U251 cells after a 1-hour incubation (FIG. 2).

Example 2 Evaluation of ST-3 Activity In Vitro Effects of ST-3 on HL60Promyelocytic Leukemia (PML) Cells and U251 GBM Cells

HL60 PML suspension cells were set at a density of 3.5×10³ cells/well in150 μL of RPMI+1.5% fetal bovine serum (FBS) in a 96 well dish. ST-3,reconstituted at a concentration of 10 mg/mL in 20 mM His, pH 7.5, wasadded to each well at a volume of 50 μL to a final concentration rangeof 0-80 μM. Cells were incubated with ST-3 for 48 hours at 37° C. Cellviability was quantified by flow cytometry using an Annexin V FITCapoptosis detection kit from Abcam. Briefly, cells were washed with PBSand resuspended in 1× assay buffer containing Annexin V FITC andpropidium iodide (PI). Annexin V detects apoptotic cells, and PI stainsdead cells. After staining, apoptotic cells show green fluorescence,dead cells show red and green fluorescence, and live cells show littleor no fluorescence. Cells were selected for analysis based on forwardscatter (FSC) vs. side scatter (SSC), and analyzed by BD Accuri C6 Plusflow cytometer to detect Annexin V-FITC binding (Ex=488 nm; Em=530 nm)using FITC signal detector and PI staining by the phycoerythrin emissionsignal detector. Percentage of Annexin V^(low) and PI^(low) werequantified and presented as % Viability. EC₅₀ values were calculatedusing GraphPad Prism v.7 XML.

Alternatively, adherent U251 or U87 cells were set at a density of3.5×10³ cells/well in 150 μL of media on day −1. On day 0, media wasremoved and replenished with 150 μL of fresh media, and cells weretreated with ST-3 as described. Following 48-hour incubation at 37° C.,floating cells were collected, adherent cells were washed withDulbecco's Phosphate Buffered Saline (DPBS), dissociated from the dishwith 50 μL 2.5% trypsin at room temperature, and combined with floatingcells. Cell viability was determined as described above for suspensioncells.

Compared to ST-2, ST-3 surprisingly demonstrates superior in vitropotency in HL60 PML cells (FIG. 3A) and in U251 GBM cells (FIG. 3B).

Effects of ST-3 on T98G GBM Cells

T98G cells were set at a density of 2×10³/well in 200 μL of RPMI+1.5%fetal bovine serum (FBS) in black-walled Nunc™ MicroWell™ 96-WellOptical-Bottom Plates with Polymer Base.

On day −1, adherent T98G cells were washed of complete media (RPMI+10%FBS) with 6 mL Dulbecco's Phosphate Buffered Saline (DPBS), anddissociated from T25 Nunclon™ Delta flask with 2 mL trypsin at roomtemperature. Trypsin was quenched with 6 mL complete media, and cellswere resuspended in fresh complete media at a concentration of 1×10⁶cells/mL. Cells were then adjusted to a concentration of 1×10⁴ cells/mLin RPMI+1.5% FBS, and 200 μL of culture was added to each well (B2 toG11) of a Nunc™ 96-well Flat Bottom Black Polystyrol Clear Bottom plateto a final cell concentration of 2×10³ cells/well. Cells were set in anincubator at 37° C. and 5% CO₂ with humidity for 24 hours.

On day 0, 100 μL of media was removed from each well. Lyophilized ST-3,freshly reconstituted in Tris Buffer (20 mM Tris+150 mM NaCl+0.05%Polysorbate-20, at pH 8.0) to stock solution of 5 mg/mL, was added toeach well at a volume of 100 μL and a final concentration range of 0-526μM. Cells were incubated with ST-3 for 72 hours prior to quantifyingcell viability.

On day 3, media was removed from wells, and wells were washed with 100μL DPBS. Cells were resuspended in 100 μL RPMI+10% FBS. CellTiter-Blue®Cell Viability Assay (Promega Corp., Madison, Wis.) was performed byadding 20 μL of CellTiter-Blue® reagent to each well. Plates werereturned to the incubator at 37° C. and 5% CO₂ with humidity for 1.5-2hours. CellTiter-Blue® assay results were read by Eppendorf PlateReaderAF2200 at Ex. 535 nm/Em. 595 nm according to manufacturer'sinstructions. Data is presented as % Viability. Cell viability wascalculated as (Sample value/Buffer control value)×100. EC₅₀ values werecalculated using GraphPad Prism v.7 XML.

ST-3 induced complete loss of cell viability, indicated by loss ofconversion of the redox dye resazurin into the fluorescent end productresorufin by viable cells. An EC₅₀ value of 69.0 μM was calculated forST-3. Results are shown in FIG. 3C. The extensive cell kill observed athigher concentrations of ST-3 indicates unexpectedly superior cytotoxicactivity over ST-2 in vitro.

This Example demonstrates that the human ATF5 bZIP domain fused to thePenetratin domain for cell entry (ST-3), is sufficient to for cytotoxicactivity in glioblastoma, neuroblastoma, and leukemia cells, and is morepotent than ST-2. A summary of the results is shown in Table 2.

TABLE 2 EC₅₀ (μM) of ATF-5-Derived Polypeptides in Cancer Cells HL60U251 T98G U87 ST-2 >200 >200 >200 >200 ST-3 16 42 69 45

Example 3 Evaluation of ST-3+ABT-263 in U87 Glioblastoma CaspaseActivation Assay

Activation of caspase enzymes occurs in the early stages of apoptosis.In this Example, we investigated the effect on caspase activation ofST-3 in combination with the Bcl-2 inhibitor ABT-263 (Navitoclax). U87cells were prepared as described in Example 2 for T98G cells.Lyophilized ST-3 was freshly reconstituted in Tris Buffer at aconcentration of 5 mg/mL, and adjusted to a working concentration of 200μM. ST-3 was added to cells in 100 μL Tris Buffer at a finalconcentration range of 0-20 μM. ABT-263 (Selleck Chem, Houston, Tex.)was reconstituted in DMSO to a concentration of 500 μM (final DMSOconcentration was 1%).

On day 0, 100 μL of media was removed from each well, followed byaddition of 98 μL of ST-3 in Tris Buffer and 2 μL of ABT-263 solution(final concentration of 0.5 μM) or DMSO control to each well.Alternatively, cells were exposed to staurosporine as positive control(10 μM).

On day 3, media was removed from wells, and wells were washed with 100μL DPBS. Cells were resuspended in 100 μL RPMI+10% FBS. Apo-ONE®Homogenous Caspase 3/7 Activity Assay (Promega Corp., Madison, Wis.) wasperformed by adding 100 of Apo-ONE® reagent to each well. Plates wereincubated at ambient temperature in the dark for 1.5-2 hours.Fluorescence was recorded by Eppendorf PlateReader AF2200 at Ex. 485nm/Em. 527 nm. Data is presented as % Caspase Activity of positivecontrol (staurosporine 10 μM). Caspase activity was calculated as(Sample value/Positive control value)×100.

Results indicate that ST-3 alone resulted in an increase in caspase 3/7activity, induced by 24-hour incubation with staurosporine (10 to28.5-38.9% of maximal response. Caspase 3/7 activity increased from15.5% with ABT-263 alone, to 50.7%, 35.7%, and 83.6% of maximal responsewhen ST-3 was administered at 5, 10, and 20 respectively, in combinationwith 0.5 μM ABT-263. Results are shown in Table 3 and FIG. 4.

TABLE 3 % Caspase Activation Conc. of ST-3 (μM) ST-3 alone ST-3 +ABT-263 0 0 15.32 5 31.48 50.76 10 28.58 35.74 20 39.11 83.60 PositiveControl 100 100

These data indicate that combination of ST-3 with ABT-263 results inenhanced activity compared to either single agent alone.

Example 4 In Vitro Activity of ST-3 Containing Lactam Bridges

We made and tested ST-3 molecules having a lactam bridge between aminoacid side chains. Positions of the bridges are shown in Table 4.

TABLE 4 Molecule Modification(s) ST-3-A K13-E20 bridge ST-3-B E25-RΔK29bridge ST-3-C AΔK26-E30 bridge ST-3-D E33-SΔK37 bridge

The Annexin V/PI cell viability assay described in Example 2 was used tomeasure cytotoxicity in HL60 cells. Addition of a lactam bridge improvedactivity of ST-3 (FIG. 5).

Example 5 In Vitro Activity of ATF5 Polypeptide with Tat CPP

We tested an ATF5 polypeptide having a Tat-derived cell penetrationdomain (SEQ ID NO: 36) fused to the ST-3 bZIP domain (SEQ ID NO: 7) inthe HL-60 cell viability assay described in Example 2. This ATF5polypeptide has cytotoxic activity, as shown in Table 5.

TABLE 5 Concentration (μM) % Viability 0 92.4 10 81.9 20 66.4 30 62.1 4054.4

Example 6 In Vivo Activity of ST-3

In this Example, we show that ST-3 affects tumor growth in multiplesubcutaneous tumor models. In the first experiment, 5×10⁶ U87 MG-Lucglioblastoma cells (MI Bioresearch) were implanted into the axilla ofNu/Nu mice. Tumors were grown to a volume of 100-200 mm³. ST-3 wasadministered at a dose of 50 mg/kg via intraperitoneal (IP) injection.The injection schedule was (QDx5, 2off)×3, followed by (Q3Dx2, 3off)×3.ST-3 treatment resulted in modest repression of tumor growth forapproximately 3 weeks (FIG. 6A).

In addition, we compared systemic administration of ST-3 via IPinjection with local administration via intratumoral (IT) injection.Subcutaneous U251 xenografts were implanted in Nu/Nu mice and grown to avolume of 100-150 mm³. ST-3 was administered Q3D at a dose of 25 mg/kgvia IP or IT injection. Tumors were excised 24 hours after the finaltreatment and processed for H&E and for TUNEL staining to detectapoptosis. Images were viewed by a pathologist and lesions werequantified. Results showed that ST-3 induced apoptosis in thesubcutaneous tumor xenograft via both systemic and local administration(Table 6).

TABLE 6 TUNEL⁺ TUNEL⁺ Treatment Route of Admin. Foci <1 mm Foci >1 mmVehicle IP 0.5 1 ST-3 IP 6 0.5 Vehicle IT 1.5 1 ST-3 IT 1 2.5* *Focimeasured 3-4 mm.

To assess the in vivo effects of ST-3 on human tumor cells, we used apatient-derived xenograft model. Briefly, adult mesenchymal GBM tumorswere passaged in mice via subcutaneous implantation into the flank.Treatment was initiated approximately 14 days post-implant. Mice wereadministered a dose of 25 mg/kg ST-3 or ST-2 IT once daily for 8 days.ST-3 delayed tumor growth better than ST-2 (FIG. 6B). Due to theaggressive nature of these tumors, a 3-7 day treatment benefit isconsidered substantial.

We also evaluated the in vivo effects of ST-3 in an HL-60 subcutaneoustumor model. Briefly, 5×10⁶ HL-60 human promyelocytic leukemia cellssuspended in 1:1 RPMI:Matrigel were injected via subcutaneous injectioninto the axilla of nu/nu mice. Beginning on day 2 following tumorinoculation, the ATF5 polypeptide was administered by subcutaneousinjection at a dose of 25 mg/kg BID for 21 days. Tumor volume (FIG. 6C)was determined over the course of the experiment.

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The present invention is further described by the following claims.

1. A cell-penetrating ATF5 polypeptide consisting essentially of acell-penetrating region and an ATF5 leucine zipper region, wherein theATF5 leucine zipper region has an amino acid sequence selected from thegroup consisting of LEGECQGLEARNRELKERAESV (SEQ ID NO: 7) andLEGECQGLEARNRELRERAESV (SEQ ID NO: 8).
 2. The cell-penetrating ATF5polypeptide according to claim 1, wherein the polypeptide comprises alactam bridge between positions of SEQ ID NO: 7 or SEQ ID NO: 8 selectedfrom the group consisting of: (a) E9 and RΔK13; (b) AΔK10 and E14; and(c) E17 and SΔK21.
 3. The cell-penetrating ATF5 polypeptide according toclaim 1, wherein the cell-penetrating region has an amino acid sequenceselected from the group consisting of: (SEQ ID NO: 20) RQIKIWFQNRRMKWKK,(SEQ ID NO: 21) RQLKLWFQNRRMKWKK, (SEQ ID NO: 35) YGRKKRRQRRR, and(SEQ ID NO: 36) YGRKKRRQRR.


4. The cell-penetrating ATF5 polypeptide according to claim 1, whereinthe polypeptide comprises an N-terminal acetyl group and/or a C-terminalamide group.
 5. The cell-penetrating ATF5 polypeptide according to claim1, which is capable of crossing the blood-brain barrier.
 6. Acomposition comprising the cell-penetrating ATF5 polypeptide accordingto claim
 1. 7. The composition according to claim 6, which is apharmaceutical composition.
 8. A kit comprising the cell-penetratingATF5 polypeptide according claim
 1. 9. A nucleic acid molecule encodingthe cell-penetrating ATF5 polypeptide according to claim
 1. 10. An invitro or ex vivo method of promoting cytotoxicity in a neoplastic cell,the method comprising contacting the neoplastic cell with thecell-penetrating ATF5 polypeptide according to claim
 1. 11. Acell-penetrating ATF5 polypeptide comprising an amino acid sequenceselected from the group consisting of: (SEQ ID NO: 3)RQIKIWFQNRRMKWKKLEGECQGLEARNRELKERAESV; (SEQ ID NO: 4)RQIKIWFQNRRMKWKKLEGECQGLEARNRELRERAESV; (SEQ ID NO: 5)YGRKKRRQRRRLEGECQGLEARNRELKERAESV; and (SEQ ID NO: 6)YGRKKRRQRRRLEGECQGLEARNRELRERAESV.


12. The cell-penetrating ATF5 polypeptide according to claim 11,comprising the amino acid sequenceRQIKIWFQNRRMKWKKLEGECQGLEARNRELKERAESV (SEQ ID NO: 3).
 13. Thecell-penetrating ATF5 polypeptide according to claim 12, wherein thepolypeptide comprises a lactam bridge at between positions selected fromthe group consisting of: (a) K13 and E20; (b) E25 and RΔK29; (c) AΔK26and E30; and (d) E33 and SΔK37.
 14. The cell-penetrating ATF5polypeptide according to claim 11, wherein the polypeptide comprises anN-terminal acetyl group and/or a C-terminal amide group.
 15. Thecell-penetrating ATF5 polypeptide according to claim 11, which iscapable of crossing the blood-brain barrier.
 16. A compositioncomprising the cell-penetrating ATF5 polypeptide according to claim 11.17. The composition according to claim 16, which is a pharmaceuticalcomposition.
 18. A kit comprising the cell-penetrating ATF5 polypeptideaccording to claim
 11. 19. A nucleic acid molecule encoding thecell-penetrating ATF5 polypeptide according to claim
 11. 20. An in vitroor ex vivo method of promoting cytotoxicity in a neoplastic cell, themethod comprising contacting the neoplastic cell with thecell-penetrating ATF5 polypeptide according to claim
 11. 21. An in vitroor ex vivo method of promoting cytotoxicity in a neoplastic cell, themethod comprising introducing into the neoplastic cell an ATF5polypeptide consisting essentially of an amino acid sequence selectedfrom the group consisting of LEGECQGLEARNRELKERAESV (SEQ ID NO: 7) andLEGECQGLEARNRELRERAESV (SEQ ID NO: 8).
 22. An in vitro or ex vivo methodof promoting cytotoxicity in a neoplastic cell, the method comprisingintroducing into the neoplastic cell a nucleic acid molecule encoding anATF5 polypeptide consisting essentially of an amino acid sequenceselected from the group consisting of LEGECQGLEARNRELKERAESV (SEQ ID NO:7) and LEGECQGLEARNRELRERAESV (SEQ ID NO: 8).